Method for determining activated coagulation factors in plasma and plasma derivatives

ABSTRACT

The invention relates to a method for determining activated coagulation factors in plasma or plasma derivatives. A sample of the plasma or plasma derivative to be examined is incubated with an activated prothrombin complex and procoagulative phospholipid vesicles, the latter containing integrated tissue factor, and thrombin formation is initiated by addition of an appropriate amount of Ca ions. After a determined incubation time, thrombin formation is terminated and the amount of thrombin formed is determined by known methods.

BACKGROUND OF THE INVENTION

The present invention relates to a method to determine activated bloodclotting factors in plasma and plasma derivatives.

When plasma is stored at low temperatures or in the course ofpurification processes, activated blood clotting factors can occur inthe plasma or in plasma derivatives. Generally, blood clotting factorsare enzymatically inactive proenzymes that are converted into theirbiologically active form through limited proteolytic splitting. Thus, atthe end of the clotting cascade thrombin (activated factor II-factorIIa), which splits fibrinogen, results from prothrombin (factor II), forexample. In addition, thrombin activates factor XIII, which bonds fibrinto a stable coagulate. In most cases, this physiological activation isdesired.

Not desired, however, is an artificial activation of blood clottingfactors in stored plasma or in the production of a purified prothrombincomplex of plasma, containing factors II, VII, IX, X as therapeuticallyeffective components. When treating patients with severe coagulationdeficiencies with prothrombin complex concentrates, e.g., through bloodloss at serious operations, thrombo-haemorrhagic complications, whosecause is unknown, may occur as undesired side effects. It is certain,however, that the risk of such complications is increased through tissuethromboplastin, which circulates in a pathologically high concentrationin the circulatory system of a patient after a severe operation. It isalso suspected that activated factors IX or X in prothrombin complexconcentrates contribute to an increased thromboses risk.

For this reason, laboratory tests are performed on the mentioned plasmasand plasma derivatives to verify the absence of activated factors. Forplasma, a method is known for selective measurement of factor VIIa inthe presence of factor VII (U.S. Pat. No. 5,472,850). In the patentpublication, an assay for factor VIIa is described which uses atruncated (shortened) tissue factor tTF. The tissue factor is anintegral membrane protein that binds both factor VII and factor VIIawith great affinity and in doing so initiates the clotting cascade. Inaddition, the complex consisting of factor VIIa and tissue factorcatalyzes the activation of factor VII to factor VIIa. The patentdescribes a mutant form of the tissue factor, whose amino acid sequenceis truncated by the portion of the protein that is responsible for thebonding to the membrane. The now soluble tissue factor tTF can bindfactor VIIa and is fully active in a coagulation test. However, it lostits capability for auto-catalysis of factor VII to factor VIIa. Thus,with the use of such a soluble mutant tissue factor in a coagulationtest, it is possible to determine selective factor VIIa in the presenceof factor VII in plasma. However, the test is relatively complicated tocarry out and offers only a result stating the presence of factor VIIa.

With prothrombin complex PPSB, a coagulation test is known and describedunder the designation NAPPT (non-activated partial prothrombin time) isstipulated as standard test (cf. H. S. KINGDON et al.; "PotentiallyThrombogenic Materials in Factor IX Concentrates"; Thromb. Diath.Haemorrh. 1975; pp. 617-631).

In this test, the clotting time of plasma is measured in the presence ofthe sample to be analyzed in the absence of substances such as kaolinthat initiate coagulation in vitro. In the absence of activatingfactors, the clotting time is about 150 sec; in the presence ofactivating factors, the clotting time is shortened. However, this testis not very sensitive and does not indicate activating factors in allinstances. A critique of this test can be found in the article by C. V.PROWSE and A. E. WILLIAMS; "A Comparison of the in vitro and in vivoThrombogenic Activity ... "; Thromb. Haemost. 1980, pp. 81-86.

SUMMARY OF THE INVENTION

This poses the task, and the present invention has the objective, ofpresenting a more precise and more sensitive test to determine thepresence of activating factors where said test presents a broad spectrumof activating factors.

This objective is achieved by a method with the following process steps:

a) A sample amount of plasma or plasma derivative is incubated withactivated prothrombin complex and procoagulating phospholipid vesicles,the latter containing integrated tissue factor, and the thrombingeneration is initiated by adding a suitable amount of Ca ions.

b) After a specified incubation time of, for example, 5 min and 10 min,the thrombin generation is ended and the amount of generated thrombin ismeasured using known methods.

Known is a test method (Y. SULTAN, F. LOYER, "In Vitro Evaluation ofFactor VIII-bypassing Activity . . . "; J. Lab. Clin, Med. 1993, p. 444pp.) where a thrombin generation is induced in the presence of a bloodplatelet suspension activated with collagen. Prior to each testformulation, blood platelets must be prepared from whole blood byrepeated centrifugation, washed and set to a specific cell number. Suchplatelet suspensions are only stable for a few hours and cannot bepreserved. This results in a costly activity that can only be carriedout by practiced operators, which limits the application of this knowntest to highly qualified and best-equipped laboratories.

The new test method, on the other hand, works with materials that can beproduced in practically unlimited amounts and that can be stored for along time such that tests can be performed without the delay due toformulating a blood platelet suspension.

The new test measures the thrombin generation in defibrinated plasma inthe presence of activated factors. The amount of generated thrombin ismeasured by applying known methods through splitting a syntheticsubstrate, for example the known S 2238 (cf. H. P. Shwarz et al.; KyotoSatellite Symposia of XIIth Congress of ISTH; Kyoto, Japan, 1989; p34-Abstract). The resultant coloration is measured photometrically.

Aside from defibrinated plasma, a test formulation also contains a smallamount of an activated prothrombin complex and procoagulatingphospholipid vesicles; with the latter containing integrated tissuefactors. The reason for selecting the presence of activated prothrombincomplex is that due to the auto-catalytic nature of the thrombingeneration with very small amounts of activated factors, the lag phaseof the thrombin generation would differ in length.

It would be impossible to specify after which incubation times tomeasure the thrombin generation. If, however, a "base noise level" isset through adding a small, specified amount of activated prothrombincomplex, this "noise level" is amplified by the activated factors. Inthis case, one can always measure after the same incubation times, forexample after 5 and 10 minutes. It should be emphasized that especiallythe use of the minimally "activated" prothrombin complex in this testand the ability to determine activated factors XII, XI, IX and VII inone test establish the particular value of the invention.

It has proven advantageous if the content of factor Xa in activatedprothrombin complex is about 1% of the total factor X content with anon-measurable content of factor IIa because this leads to the bestreproducible "base noise level".

The tissue factor to be used is produced from biogenic startingmaterial, where particularly dry acetone powder is extracted from bovinebrain with a detergent-containing solution.

For integration in procoagulating phospholipid vesicles indetergent-containing solution, the tissue factor is mixed withphosphatidylserine and phosphatidylcholine and integrated in theprocoagulating phospholipid vesicles by dialysis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Exemplary embodiments of the invention are explained in connection withthe following examples:

Example 1:

1. Production of Defibrinated Plasma

Ancrod (the poison of the snake species Ankistrodon rhodostoma; SigmaChemical C.: Art. No. A 5042) is diluted to 10 U/ml using distilledwater. 1 ml of human plasma from normal donators is mixed with 10 μl ofAncrod solution, incubated for 1 hour at 37° C., quickly chilled to -70°C., reheated to 37° C. and centrifuged. The supernatant, defibrinatedplasma, is stored at room temperature and must be used within 10minutes. The defibrinated plasma can also be portioned and frozen. Itcontains only negligible traces if thrombin.

2. Production of the Partial Prothrombin Complex (PPC)

Required Reagents:

standard plasma

DEAE-Sephadex A 50

elution solution: 30 g NaCl per liter of distilled water wash buffer. 9g Na₂ HPO₄.2 H₂ O+7 g NaCl per liter distilled water, pH adjusted to7.0;

equilibration buffer. 6 g Na₃ -citrate.2 H₂ O+7 g NaCl per literdistilled water, pH not adjusted.

50 ml normal plasma is frozen, defrosted overnight at 4° C. andcentrifuged for 15 min. The supernatant is "cryogenic supernatant"; 0.5units of heparin are added per ml cryogenic supernatant to the latter.

DEAE-Sephadex is swelled for 15 minutes in 1 M NaCl solution at roomtemperature (21-24° C.) or overnight at 4° C. (20 mg dry mass to 2 mlsolution), re-suspended by repeated filtration through a nylon net andequilibrated in an equilibration buffer. Heparin-binding proteins areabsorbed from the cryogenic supernatant to the ion exchanger byincubation at 4° C. for one hour. In this manner 40 ml cryogenicsupernatant is added to equilibrated DEAE-Sephadex corresponding to 20mg dry mass. The loaded DEAE-Sephadex is filtered and purified in washbuffer through re-suspending. After renewed filtering, the DEAE-Sephadexis suspended in 1 ml of elution buffer and shaken at room temperaturefor 15 minutes.

After removing the DEAE-Sepahdex, the solution of the partialprothrombin complex is dialyzed over night against distilled waterpre-cooled to 4° C. and frozen in portions. 3. Production of theActivated Prothrombin Complex (APC)

A suitable dilution of the PPC according to point 2 must be detected inthe thrombin generation test (see point 5). For this purpose, a dilutionseries is made using HEPES buffer (e.g., 1:20, 1:40 and 1:80;HEPES=N-(2hydroxyethyl)piperazin-N'-(2-ethane sulphonic acid). Eachdilution is incubated for 5 minutes at 37° C. with CaCl₂ in a finalconcentration of 10 mM. A suitable dilution is characterized by thelinearity of the speed of splitting of the chromogenic substrate S 2238.The ready-to-use APC can be frozen in portions. Using this procedure, areproducible APC can be produced with a factor Xa content of about 1% ofthe total factor X in the preparation. After incubation of the APC,thrombin is not detectable with the chromogenic substrate S 2238.

4. Manufacture of Procoagulating Phospholipid Vesicles With IntegratedTissue Factor

Required Reagents:

dialysis hose, cellulose membrane of Sigma (item no. D 9402) orSlide-A-Lyzer™ dialysis cassette from Pierce;

dry acetone powder from bovine brain (Brain Acetone Powder, Sigma, itemno. B 0508);

n-octyl-β-D-glucopyranoside (short, octyl glucoside) from Alexis (itemno. 500-001-G005); 100 mg/ml distilled water (10% G/V solution);

phosphatidylserine (PS), (Sigma, item no. P 1185)

phosphatidylcholine (PC), (Sigma, item no. P 4139). Both phospholipidsmay contain unsaturated but preferably saturated fatty acids;

dialysis buffer in the form of 94.5 g saccharose and 0.27 g NaCl perliter of distilled water. The dialysis buffer may also contain 0.5%NaN₃. 0.216 g dry acetone powder is digested with 1 ml of 10%octylglucoside solution at about 30° C. in an ultrasound bath undershaking. The insoluble portion is removed by centrifuging; per ml ofsupernatant, 20 mg phospholipid solution (mole ratio PC to PS 6 to 4) isdissolved and dialyzed against the dialysis buffer. With daily change ofthe dialysis buffer, dialysis is carried out for 3 days, then two moredays against a dialysis buffer that contains a gel for bindingdetergents (e.g., Biorad SM-2). The detergent is removed throughdialysis, and a suspension of vesicles is generated spontaneously thatcontains the tissue factor in an integrated manner. The obtainedvesicles can be frozen in portions.

With each new preparation, a suitable dilution is searched for using theblind value in the thrombin generation test (see point 5), where theblind value shall be a delta OD of 0.005 to 0.010.

5. Thrombin Generation Test

Required Reagents:

HEPES/NaCl buffer. 6 mM HEPES, 139 mM NaCl, 3.5 g albumin per 1 buffer,pH 7.35;

citrate buffer: 20 mM Na₃ -citrate.2 H₂ O, 125 mM NaCl, pH 7.35;

starting solution 375 mM CaCl₂, stop solution 40 mM EDTA;

APC and vesicle (point 4) with integrated tissue factor in a suitabledilution 5.1 Test of the plasma for a presence of activated clottingfactors

In a known manner 1% Triton X-100 and 1% Tributyl phosphate is added topooled plasma made either of whole blood or through plasmaphoresis toinactivate membrane-sheathed viruses (Neurath, A. R. and Horowitz, B.,EP-PS 131 740) and incubated for 4 hours at 30° C.). Thereafter, the twochemicals are removed in a known manner through hydrophobicchromatography (Bonomo, R. J., EP-PS 366 946). To prepare for the testfor present activated clotting factors, a plasma sample is difibrinatedas described in example 1.

Pipetting Scheme

for the blind value: 250 μl HEPES/NaCl buffer+250 μl defibrinated plasma(point 1);

for the APC: 250 μl HEPES/NaCl buffer+250 μl defibrinated plasma+125 μlAPC;

for the plasma sample: 125 μl HEPES/NaCl buffer+125 μl defibrinatedplasma+125 μl APC+125 μl defibrinated plasma sample of the plasma thatis to be analyzed for activated factors.

50 μl citrate buffer, 140 μl HEPES/NaCl buffer and 10 μl phospholipidsuspension, made according to point 4, is added to each formulation andincubated for 5 minutes at 37° C. The reaction is started with 10 μlCaCl₂ solution, after 5 minutes and 10 minutes, 50 μl of the mixture isadded each time to 50 μl of a predefined EDTA solution (EDTA=ethylenediamine tetraacidic acid) to stop the thrombin generation.

6. Test For Thrombin Activity

The method is basically know and is supplied as a set. It is onlymentioned for the sake of completeness.

Required Reagents:

Chromogenic substrate S 2238, Chromogenix. item no. 41202. The contentof one bottle of S 2238 (25 mg) is dissolved in 20 ml of distilledwater; a working dilution of 1 mM is produced by diluting with distilledwater at a ratio of 1:1. Thrombin, 53 nkat, Chromogenix, item no. 41217,is dissolved in 1 ml distilled water. A buffer concentrate of 500 mMTris, 75 mM EDTA, 10% G/V PEG 6000, pH 8.4, is diluted with distilledwater 1:10. A working buffer is made by mixing buffer concentrate (30ml) with human serum albumin (HSA), 20% HSA (0.75 ml). From this, asubstrate buffer is mixed, containing 26 ml working buffer and 2.4 mlsubstrate solution.

20 μl of the sample amounts, called "pipetting scheme" under point 5.1,are each mixed with 500 μl substrate buffer that has been preheated to37° C. The extinction is measured at one-minute intervals. To controlthe linearity of substrate splitting, 5 extinction cycles of 1 minuteeach are measured at 405 nm. A calibration line is established bydiluting thrombin with working buffer in the range of 5.3 to 0.53 nkat.

The following results were achieved with virus-inactivated plasmasamples, as produced in point 5:

1,2,3 . . . different production batches of virus-inactivated plasma

    ______________________________________                                        B*     APC       1      2        3    4                                       ______________________________________                                        0.003  0.028     0.036  0.035    0.035                                                                              0.033                                   5      6         7      8        9    10                                      ______________________________________                                        0.041  0.038     0.057  0.045    0.060                                                                              0.051                                   ______________________________________                                         B* blind value                                                           

The numbers presented are extinction changes per minute and have beenobtained after an incubation of 10 minutes at the thrombin generationtest. The measurement at 5 minutes incubation time provides no or verysmall extinction changes.

To evaluate for thrombin generation from activated clotting factors, thevalues for APC and the blind value are subtracted from the measuredextinction difference per minute. In the example of batch 9, thisresults in a value of 0.029. At a calibration line with thrombindilution, a thrombin activity reading of 2 nkat is obtained.

If plasma samples contain heparin, the heparin must be neutralized priorto the test with protamine sulfate or Polybren™ (hexadimethrine bromide)or preferably inactivated with heparinase.

Example 2--Detection of Activated Factor IX, Factor XI and Factor XII:

Factor IXa, factor XIa and factor XIIa have been obtained from Alexis,items no. 73510-1, 200-039-C025 and 200-042-UC02. Defibrinated plasmahas been activated through incubation with Thrombin (1.66 nkat, 10 minat 37° C.), thereafter, the thrombin activity was inhibited withPefabloc SC™ (4-(2-aminoethyl)-benzo sulphonyl fluoride hydrochloride),Boehringer Mannheim, item no. 1 429 868. The amount of Pefabloc SC™required to inhibit the thrombin activity at hand has been determined ina pretest.

Pipetting Scheme:

blind value: 250 μl HEPES/NaCl buffer+250 μl defibrinated plasma;

APC: 125 μl APC+125 μl HEPES/NaCl buffer+250 μl defibrinated plasma;

factor IXa, 10 μl of a solution with 0.2 plasma units or 10 μl factorXIa or 10 μl factor XIIa+125 μl APC+115 μl HEPES/NaCl buffer+250 μldefibrinated plasma.

Thereafter, 50μl citrate buffer, 140 μl HEPES/NaCl buffer and 10 μlphospholipid suspension, made according to example 1, point 4, is addedto each formulation and incubated for 5 minutes at 37 °0C. The reactionis started with 10 μl CaCl₂ solution, after 5 minutes and 10 minutes, 50μl of the mixture is added each time to 50 μl of a predefined EDTAsolution to stop the thrombin generation.

    ______________________________________                                                    F IXa + F XIa +                                                                             F XIIa +                                                                             Incubation time in                           B*   APC    APC     APC   APC    thrombin generation test                     ______________________________________                                        0.003                                                                              0.006  0.130   0.034 0.052   5 min                                       0.022                                                                              0.039  0.122   0.083 0.088  10 min                                       ______________________________________                                         *blind value                                                             

The manufacturer of factor IXa, factor XIa and factor XIIa only liststhe activity in plasma units for factor IXa, where one plasma unitcorresponds to the amount of the factor in 1 ml standard plasma. Usingthe pipetting scheme and the test process, one can calculate that in thetest 0.003 plasma units factor IXa initiate a clearly measurablethrombin generation after an incubation time of only 5 minutes.

Example 3--Detection of Activated Factor VII:

Factor VIIa has been obtained from Alexis, item no. 73560-1 or HF086-1.Defibrinated plasma was not activated with thrombin. Pipetting schemeand test procedure were the same as stated in example 2. In the thrombingeneration test with factor VIIa, phospholipid vesicles with and withoutintegrated tissue factor as cofactor were analyzed in direct comparison.Vesicles without tissue were used in undiluted form, while vesicles withintegrated tissue factor had to be pre-diluted by a factor of 2000. Thefollowing results were obtained in such a comparison:

    ______________________________________                                                            F VIIa + incubation time in the                                   B*   APC    APC      thrombin generation test                         ______________________________________                                        vesicles without                                                                        0.003  0.026  0.035  10 min                                         tissue factor                                                                 vesicles with                                                                           0.015  0.039  0.061  10 min                                         tissue factor                                                                 ______________________________________                                         *blind value                                                             

With both test series, there were 0.002 plasma units factor VIIa in thetest. In a test series with vesicles with integrated tissue factor,0.001 plasma units factor VIIa were still clearly detectable.

There has thus been shown and described a novel method to determineactivated blood clotting factors in plasma and plasma derivatives whichfulfills all the objects and advantages sought therefor. Many changes,modifications, variations and other uses and applications of the subjectinvention will, however, become apparent to those skilled in the artafter considering this specification and the accompanying drawings whichdisclose the preferred embodiments thereof. All such changes,modifications, variations and other uses and applications which do notdepart from the spirit and scope of the invention are deemed to becovered by the invention, which is to be limited only by the claimswhich follow.

What is claimed is:
 1. A method for determining activated clottingfactors in plasma and plasma derivatives, using the following processingsteps:a) incubating a sample amount of plasma or plasma derivative withactivated prothrombin complex and procoagulating phospholipid vesicles,the latter containing integrated tissue factor, and the thrombingeneration is initiated by adding a suitable amount of Ca²⁺ ions, b)after a specified incubation time of 5 min and 10 min, the thrombingeneration is ended and the amount of generated thrombin is measuredusing known methods.
 2. The method set forth in claim 1, wherein thecontent of factor Xa in the activated prothrombin complex is about 1% ofthe total factor X content with a non-measurable content of factor IIa.3. The method set forth in claim 1, wherein the tissue factor isproduced from biogenic starting material.
 4. The method set forth inclaim 3, wherein the tissue factor is extracted from bovine brain with adetergent-containing solution of dry acetone powder.
 5. The method setforth in claim 1, wherein the tissue factor is integrated inprocoagulating phospholipid vesicles.
 6. The method set forth in claim4, wherein the tissue factor is mixed in a detergent-containing solutionwith phosphatidylserine and phosphatidylcholine and integrated in theprocoagulating phospholipid vesicles by dialysis.
 7. The method setforth in claim 5, wherein the tissue factor is mixed in adetergent-containing solution with phosphatidylserine andphosphatidylcholine and integrated in the procoagulating phospholipidvesicles by dialysis.